Totally Accellent siRNAs

This time last year, Thermo Fisher's Dharmacon group was announcing a breakthrough in reducing off-target events when using short interfering RNAs (siRNA) by chemically modifying the guide strand to silence off-target transcripts.

This year, it has gone one better, launching a new range of modified siRNAs and an optimised delivery media that it says can effectively silence genes in all cell types tested to date.

One of the major problems of RNAi research, besides the off-target events, is actually getting siRNAs into difficult to transfect cells such as primary cells, stem cells and neurons. What Thermo Fisher has done with its new range of Dharmacon Accell siRNAs is enable them to be absorbed directly by the cells without having to use transfection agents, viruses or electroporation.

"What we see with lipid delivery, which is probably most common, is that they are only applicable to certain cell types," Thermo Fisher's director of biology R&D for Dharmacon products, Dr Devin Leake, says.

"There are many difficult-to-transfect cells that are not amenable to lipid delivery methods - these would be immune cells, Jurkats and primary cells. For researchers who want to work with the most relevant cell types possible they had to switch to other types of technology such as viral."

For viral delivery, Thermo Fisher has released its Dharmacon SMARTvector shRNA lentiviral particles and sees Accell as a complementary technology.

"With viral work, specifically with lentivirus, you have constructs that integrate and result in long-term expression of shRNA," Leake says. "With Accell you have transient silencing. However, the other amazing thing about Accell is that you can get it into difficult-to-transfect cell types such as the Jurkats and the primary cells, and you can deliver several doses over a period of time for a longer duration of silencing.

"We've seen examples where you can have an Accell siRNA providing sustained knock-down for as much as a month. If your assay is such that you need several months then shRNA and viral constructs are ideal, but if you need transient knock-down, then Accell allows you to get into any cell type that you have."

Leake also says that as Accell is absorbed into the cell, it provides a very gentle delivery method that lacks the stress effects seen with lipid delivery.

"We also don't see the off-target effects that typically plague unmodified siRNAs," he says. "So we get all of the benefits of the delivery and specificity.

"Because you are not formulating the siRNA the protocol is much easier, and as such it is more applicable to all researchers, not just the RNAi researchers. Now, anyone can use RNAi - you take resuspend the siRNA, you add it to your cells and you're done."

Thermo Fisher's release of Dharmacon Accell comes at a very interesting time in RNAi research, following a fascinating paper published in Nature in March (doi:10.1038/nature06765) by a team of researchers at the University of Kentucky, led by a professor of ophthalmology, Jayakrishna Ambati, looking at the use of RNAi in macular degeneration.

Two US biotechs, Sirna Therapeutics and OPKO, are in the midst of clinical trials using siRNAs to turn off the vascular endothelial growth factor-A (VEGFA) or its receptor in patients with blinding choroidal neovascularisation.

Ambati's paper suggests that in this instance, the siRNAs are not actually penetrating the cell but are binding to the receptor, and as such are working in an untargeted sense. The paper also suggests that siRNAs may induce a pro-inflammatory response through toll-like receptor 3 (TLR3).

Leake says the paper is interesting. "I think that there are siRNA sequence motifs that do elicit a pro-inflammatory response. What is surprising to me, is that modifications do not reduce the toll receptor response.

"Although we haven't looked at toll 3 receptor, we have looked at the downstream outputs that are described - IL12 response and the interferon gamma response - and when modifications are incorporated into siRNAs, we don't see a pro-inflammatory response. While I believe the results presented in the paper, the effect may not be broadly applicable to other siRNA sequences.